Polyclonal populations of bispecific molecules and methods of production and uses thereof

Abstract

The invention relates to a polyclonal population of bispecific molecules which comprises a plurality of different bispecific molecules, each comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a pathogenic antigenic molecule such that the plurality of different bispecific molecules have different second antigen recognition portions with different recognition specificities, such as with recognition specificities directed to different epitopes and/or different variants of a pathogen and/or to different pathogens. The invention also relates to methods of producing such polyclonal population of bispecific molecules. The invention further relates to methods of using such polyclonal population of bispecific molecules for the clearance of pathogens from the circulatory system of a mammal.

What is claims is:  
     
         1 . A polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a pathogenic antigenic molecule.  
     
     
         2 . The polyclonal population of bispecific molecules of  claim 1 , wherein said first antigen recognition portion comprises an anti-CR1 monoclonal antibody.  
     
     
         3 . The polyclonal population of bispecific molecules of  claim 1 , wherein said plurality of bispecific molecules comprises different bispecific molecules comprising different first antigen recognition portions.  
     
     
         4 . The polyclonal population of bispecific molecules of  claim 1 , wherein said second antigen recognition portion comprises a monoclonal antibody.  
     
     
         5 . The polyclonal population of bispecific molecules of  claim 1 , wherein said plurality of bispecific molecules comprises bispecific molecules comprising different second antigen recognition portions having different antigen recognition specificities directed to different epitopes on a pathogen.  
     
     
         6 . The polyclonal population of bispecific molecules of  claim 1 , wherein said plurality of bispecific molecules comprises bispecific molecules comprising different second antigen recognition portions having different antigen recognition specificities directed to different variants of a pathogen.  
     
     
         7 . The polyclonal population of bispecific molecules of  claim 1 , wherein said plurality of bispecific molecules comprises bispecific molecules comprising different second antigen recognition portions having different antigen recognition specificities directed to different pathogens.  
     
     
         8 . The polyclonal population of bispecific molecules of  claim 5 , comprising bispecific molecules that are synergistic in neutralization of said pathogen.  
     
     
         9 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of bispecific molecules constitutes at least 10% of said population.  
     
     
         10 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of bispecific molecules constitutes at least 20% of said population.  
     
     
         11 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of bispecific molecules constitutes at least 50% of said population.  
     
     
         12 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of bispecific molecules constitutes at least 80% of said population.  
     
     
         13 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of bispecific molecules constitutes at least 90% of said population.  
     
     
         14 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein no single bispecific molecule has a proportion exceeding 95% of said plurality.  
     
     
         15 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein no single bispecific molecule has a proportion exceeding 80% of said plurality.  
     
     
         16 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein no single bispecific molecule has a proportion exceeding 60% of said plurality.  
     
     
         17 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein no single bispecific molecule has a proportion exceeding 50% of said plurality.  
     
     
         18 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of different bispecific molecules comprises at least 2 different bispecific molecules having different second antigen recognition portions.  
     
     
         19 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of different bispecific molecules comprises at least 10 different bispecific molecules having different second antigen recognition portions.  
     
     
         20 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said plurality of different bispecific molecules comprises at least 100 different bispecific molecules having different second antigen recognition portions.  
     
     
         21 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said population is purified.  
     
     
         22 . The polyclonal population of bispecific molecules of any one of claims  1 - 8 , wherein said second antigen recognition portions are from a hyperimmune serum.  
     
     
         23 . A polyclonal population of bispecific molecules comprising a plurality of bispecific molecules that are produced by cross-linking a plurality of first antigen recognition portions that bind C3b-like receptors and a plurality of different second antigen recognition portions that bind pathogenic antigenic molecules.  
     
     
         24 . The polyclonal population of bispecific molecules of  claim 23 , wherein said plurality of different second antigen recognition portions is from a hyperimmune serum.  
     
     
         25 . A method for producing a polyclonal population of bispecific molecules, comprising cross-linking a plurality of first antigen recognition portions that bind C3b-like receptors and a plurality of different second antigen recognition portions that bind pathogenic antigenic molecules.  
     
     
         26 . A method for producing a polyclonal population of bispecific molecules, comprising: 
 (a) producing a plurality of first antigen recognition portions that bind a C3b-like receptor;    (b) producing a plurality of different second antigen recognition portions that bind one or more pathogenic antigenic molecules; and    (c) cross-linking said plurality of first antigen recognition portions and said plurality of different second antigen recognition portions, to produce said polyclonal population of bispecific molecules.    
     
     
         27 . The method of  claim 25  or  26 , wherein said plurality of different second antigen recognition portions is from a hyperimmune serum.  
     
     
         28 . The method of  claim 25  or  26 , wherein said plurality of different second antigen recognition potions is obtained from a phage display library.  
     
     
         29 . The method of  claim 28 , wherein said plurality of second antigen recognition portions is produced by a method comprising expressing in a host a plurality of eukaryotic expression vectors each containing a nucleotide sequence encoding the heavy and light chain variable regions of a different immunoglobulin in a polyclonal library of immunoglobulin molecules, wherein said nucleotide sequences encoding the heavy and light chain variable regions comprise nucleotide sequences encoding the respective heavy and light chains linked head to head to form a bidirectional vector.  
     
     
         30 . The method of  claim 28 , wherein said plurality of second antigen recognition portions is produced by a method comprising: 
 (a) selecting from a phage display library a plurality of phages that display antigen recognition polypeptides having different respective binding specificities to one or more antigenic molecules using affinity screening;    (b) obtaining a plurality of nucleic acids encoding said plurality of antigen recognition polypeptides, respectively; and    (c) expressing said plurality of nucleic acids in a host, to produce said polyclonal population of second antigen recognition portions.    
     
     
         31 . The method of  claim 25  or  26 , wherein said cross-linking comprises using cross-linking agents N-succinimidyl S-acetylthioacetate and sulfosuccinimidy 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.  
     
     
         32 . The method of  claim 25  or  26 , wherein said cross-linking comprises using cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP).  
     
     
         33 . A method for producing a synergistic polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds an epitope of the same pathogen or pathogenic antigenic molecule, said method comprising combining a polyclonal population of bispecific molecules with one or more monoclonal bispecific molecules that show synergistic activities in neutralization of said pathogen or pathogenic antigenic molecule when combined with said polyclonal population, to produce a synergistic polyclonal population of bispecific molecules.  
     
     
         34 . A method for producing a synergistic polyclonal population of bispecific molecules, comprising: 
 (a) producing a plurality of first antigen recognition portions that bind C3b-like receptors;    (b) producing a plurality of different second antigen recognition portions that bind the same pathogen or pathogenic antigenic molecule;    (c) cross-linking said plurality of first antigen recognition portions and said plurality of different second antigen recognition portions to produce a polyclonal population of bispecific molecules;    (d) obtaining one or more monoclonal second antigen recognition portions that are synergistic in neutralization of said pathogen or pathogenic antigenic molecule;    (e) cross-linking said first antigen recognition portion and said one or more monoclonal second antigen recognition portions to produce one or more monoclonal bispecific molecules; and    (f) incorporating in said polyclonal population of bispecific molecules said one or more monoclonal bispecific molecules, to produce a synergistic polyclonal population of bispecific molecules.    
     
     
         35 . A method of treating a mammal having a disease or disorder or undesirable condition associated with the presence of one or more pathogens or pathogenic antigenic molecules, comprising administering to said mammal a therapeutically effective dose of a polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds an epitope of said one or more pathogens or pathogenic antigenic molecules.  
     
     
         36 . A method of preventing a disease or disorder or undesirable condition associated with the presence of one or more pathogens or pathogenic antigenic molecules in a mammal, comprising administering prior to the onset of said disease or disorder or undesirable condition, to said mammal a prophylactically effective dose of a polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds an epitope of said one or more pathogens or pathogenic antigenic molecule.  
     
     
         37 . A method of treating a mammal having a disease or disorder or undesirable condition associated with the presence of one or more pathogens or pathogenic antigenic molecules, comprising administering to said mammal a therapeutically effective dose of a polyclonal population of bispecific molecules and one or more monoclonal bispecific molecules, said polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a pathogenic antigenic molecule, said monoclonal bispecific molecules are synergistic in neutralization of said one or more pathogens or pathogenic antigenic molecules when combined with said polyclonal population.  
     
     
         38 . The method of  claim 35 ,  36  or  37 , wherein said administering is intravenous.  
     
     
         39 . The method of  claim 35 ,  36  or  37 , wherein said mammal is a human, and said C3b-like receptor is CR1.  
     
     
         40 . The method of  claim 35 ,  36  or  37 , wherein said mammal is a non-human mammal.  
     
     
         41 . The method of  claim 35 ,  36  or  37 , wherein said pathogens comprise an autoimmune antigen.  
     
     
         42 . The method of  claim 35 ,  36  or  37 , wherein said pathogens comprise an infectious agent.  
     
     
         43 . The method of  claim 42 , wherein said infectious agent is a virus.  
     
     
         44 . The method of  claim 43 , wherein said virus is HIV-1 virus.  
     
     
         45 . The method of  claim 42 , wherein said infectious agent is a bacterium.  
     
     
         46 . The method of  claim 42 , wherein said infectious agent is a fungus.  
     
     
         47 . The method of  claim 42 , wherein said infectious agent is a parasite.  
     
     
         48 . The method of  claim 47 , wherein said parasite is a protozoan.  
     
     
         49 . The method of  claim 35 ,  36  or  37 , wherein said pathogens comprise a poisonous compound.  
     
     
         50 . The method of  claim 49 , wherein said poisonous compound is a poisonous compound contained in a venom.  
     
     
         51 . The method of  claim 50 , wherein said venom is reptile venom.  
     
     
         52 . The method of  claim 51 , wherein said venom is snake venom.  
     
     
         53 . The method of  claim 51 , wherein said venom is insect venom.  
     
     
         54 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules comprises bispecific molecules comprising different second antigen recognition portions having different antigen recognition specificities directed to different epitopes of the same pathogen or pathogenic antigenic molecule.  
     
     
         55 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules comprises bispecific molecules comprising different second antigen recognition portions having different antigen recognition specificities directed to different variants of a pathogen.  
     
     
         56 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules comprises bispecific molecules comprising different second antigen recognition portions having different antigen recognition specificities directed to different pathogens.  
     
     
         57 . The method of  claim 35 ,  36  or  37 , wherein said plurality of different bispecific molecules comprising at least 2 different bispecific molecules having different second antigen recognition portions.  
     
     
         58 . The method of  claim 35 ,  36  or  37 , wherein said plurality of different bispecific molecules comprises at least 10 different bispecific molecules having different second antigen recognition portions.  
     
     
         59 . The method of  claim 35 ,  36  or  37 , wherein said plurality of different bispecific molecules comprises at least 100 different bispecific molecules having different second antigen recognition portions.  
     
     
         60 . The method of  claim 35 ,  36  or  37 , wherein said first antigen recognition portion comprises an anti-CR1 monoclonal antibody.  
     
     
         61 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules comprises different bispecific molecules comprising different first antigen recognition portions.  
     
     
         62 . The method of  claim 35 ,  36  or  37 , wherein said second antigen recognition portion comprises a monoclonal antibody.  
     
     
         63 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules constitutes at least 10% of said population.  
     
     
         64 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules constitutes at least 20% of said population.  
     
     
         65 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules constitutes at least 50% of said population.  
     
     
         66 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules constitutes at least 80% of said population.  
     
     
         67 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules constitutes at least 90% of said population.  
     
     
         68 . The method of  claim 35 ,  36  or  37 , wherein said plurality of bispecific molecules constitutes at least 95% of said population.  
     
     
         69 . The method of  claim 35 ,  36  or  37 , wherein no single bispecific molecule has a proportion exceeding 80% of said plurality.  
     
     
         70 . The method of  claim 35 ,  36  or  37 , wherein no single bispecific molecule has a proportion exceeding 60% of said plurality.  
     
     
         71 . The method of  claim 35 ,  36  or  37 , wherein no single bispecific molecule has a proportion exceeding 50% of said plurality.  
     
     
         72 . A kit comprising in a container a polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a pathogenic antigenic molecule.  
     
     
         73 . A kit comprising in two or more containers a) a polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a pathogenic antigenic molecule; and b) one or more monoclonal bispecific molecules each comprising a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds said pathogenic antigenic molecule, said one or more monoclonal bispecific molecules are synergistic with said plurality in neutralization of said pathogenic antigenic molecule.  
     
     
         74 . A kit comprising in two or more containers a) a polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a pathogenic antigenic molecule; and b) one or more monoclonal antibodies each comprising an antigen recognition portion that binds said pathogenic antigenic molecule, said one or more monoclonal antibodies are synergistic with said plurality in neutralization of said pathogenic antigenic molecules.  
     
     
         75 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of different bispecific molecules comprises at least 2 different bispecific molecules having different second antigen recognition portions.  
     
     
         76 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of different bispecific molecules comprises at least 10 different bispecific molecules having different second antigen recognition portions.  
     
     
         77 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of different bispecific molecules comprises at least 100 different bispecific molecules having different second antigen recognition portions.  
     
     
         78 . The kit of  claim 72 ,  73  or  74 , wherein said first antigen recognition portion in said plurality comprises an anti-CR1 monoclonal antibody.  
     
     
         79 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of bispecific molecules comprises different bispecific molecules comprising different first antigen recognition portions.  
     
     
         80 . The kit of  claim 72 ,  73  or  74 , wherein said second antigen recognition portion in said plurality comprises a monoclonal antibody.  
     
     
         81 . The kit of  claim 80 , wherein said plurality of bispecific molecules comprises bispecific molecules comprising different second antigen recognition portions having different antigen recognition specificities directed to different epitopes of the same pathogen.  
     
     
         82 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of bispecific molecules constitutes at least 10% of said population.  
     
     
         83 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of bispecific molecules constitutes at least 20% of said population.  
     
     
         84 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of bispecific molecules constitutes at least 50% of said population.  
     
     
         85 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of bispecific molecules constitutes at least 80% of said population.  
     
     
         86 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of bispecific molecules constitutes at least 90% of said population.  
     
     
         87 . The kit of  claim 72 ,  73  or  74 , wherein said plurality of bispecific molecules constitutes at least 95% of said population.  
     
     
         88 . The kit of  claim 72 ,  73  or  74 , wherein no single bispecific molecule has a proportion exceeding 80% of said plurality.  
     
     
         89 . The kit of  claim 72 ,  73  or  74 , wherein no single bispecific molecule has a proportion exceeding 60% of said plurality.  
     
     
         90 . The kit of  claim 72 ,  73  or  74 , wherein no single bispecific molecule has a proportion exceeding 50% of said plurality.  
     
     
         91 . A population of modified hematopoietic cells that consists essentially of a population of hematopoietic cells each bound to one or more bispecific molecules, wherein each of said bispecific molecules comprises a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a pathogenic antigenic molecule, wherein said bispecific molecules bound to said population of modified hematopoietic cells forms a population of bispecific molecules comprising different second antigen recognition portions.  
     
     
         92 . A polyclonal population of bispecific molecules comprising a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different immunoglobulin molecule that binds a pathogen or pathogenic antigenic molecule.  
     
     
         93 . The polyclonal population of bispecific molecules of  claim 92 , wherein said first antigen recognition portion comprises an anti-CR1 monoclonal antibody.  
     
     
         94 . The polyclonal population of bispecific molecules of  claim 92 , wherein said plurality of bispecific molecules comprises different bispecific molecules comprising different first antigen recognition portions.  
     
     
         95 . The polyclonal population of bispecific molecules of  claim 92 , wherein said different immunoglobulin molecules have different antigen recognition specificities directed to different epitopes of the same pathogen or pathogenic antigenic molecule.  
     
     
         96 . The polyclonal population of bispecific molecules of  claim 92 , wherein said different immunoglobulin molecules have different antigen recognition specificities directed to different variants of a pathogen or pathogenic antigenic olecule.  
     
     
         97 . The polyclonal population of bispecific molecules of  claim 92 , wherein said different immunoglobulin molecules have different antigen recognition specificities directed to different pathogens or pathogenic antigenic molecules.  
     
     
         98 . The polyclonal population of bispecific molecules of  claim 92 , comprising bispecific molecules that are synergistic in neutralization of said pathogen or pathogenic molecule.  
     
     
         99 . The polyclonal population of bispecific molecules of any one of claim  92 - 98 , wherein said plurality of bispecific molecules constitutes at least 10% of said population.  
     
     
         100 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said plurality of bispecific molecules constitutes at least 20% of said population.  
     
     
         101 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said plurality of bispecific molecules constitutes at least 50% of said population.  
     
     
         102 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said plurality of bispecific molecules constitutes at least 80% of said population.  
     
     
         103 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said plurality of bispecific molecules constitutes at least 90% of said population.  
     
     
         104 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein no single bispecific molecule has a proportion exceeding 95% of said plurality.  
     
     
         105 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein no single bispecific molecule has a proportion exceeding 80% of said plurality.  
     
     
         106 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein no single bispecific molecule has a proportion exceeding 60% of said plurality.  
     
     
         107 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein no single bispecific molecule has a proportion exceeding 50% of said plurality.  
     
     
         108 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said plurality of different bispecific molecules comprises at least 2 different bispecific molecules.  
     
     
         109 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said plurality of different bispecific molecules comprises at least 10 different bispecific molecules.  
     
     
         110 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said plurality of different bispecific molecules comprises at least 100 different bispecific molecules.  
     
     
         111 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said population is purified.  
     
     
         112 . The polyclonal population of bispecific molecules of any one of claims  92 - 98 , wherein said bispecific molecules in said plurality comprise immunoglobulin molecules obtained from the same hyperimmune serum.